Conducting polymers and polymer-biological tissue composites for tissue growth and regeneration

ABSTRACT

Conjugated, electrically conducting polymers (CPs) with the ability to covalently graft onto collagen and collagenic materials are provided. Also provided are methods of functionalizing biological tissues and other biological substrates with the CPs, and methods of using the functionalized biological substrates as cell and tissue growth scaffolds that harness the passive therapeutic benefits of CPs and use the enhanced conductivity provided by the scaffolds to stimulate cell growth and proliferation through the bulk of the biological substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication No. 63/010,181 that was filed Apr. 15, 2020, and U.S.provisional patent application No. 63/010,156 that was filed Apr. 15,2020, the entire contents of which are incorporated herein by reference.

BACKGROUND

Incorporation of electroactive elements, including conducting polymers,graphene, and carbon nanotubes in a biomaterial has a significant effecton cellular adhesion, proliferation, and differentiation, as well asallowing for both the possibility of electrical stimulation and sensingto both augment and better understand the environment of regeneratingtissue. However, achieving a complex three-dimensional (3D) structure,biologically relevant mechanical properties, and cell binding domainsalong with the electroactive element is an ongoing challenge. Complexcomposites of synthetic and/or natural polymers with conductingelements, electrospun fibers coated with conducting elements, or 3Dprinting and bioprinting have been proposed. However, at best, theseapproaches achieve a “compromise” material that is generally lacking inone of the three aforementioned attributes.

SUMMARY

Biocompatible electronically conductive polymers and biologicalsubstrates funcationalized with the electronically conductive polymersare provided. Also provided are methods of functionalizing thebiological substrates and methods of using the functionalized biologicalsubstrates in tissued engineering applications, including cell cultures.

One embodiment of a biocompatible electronically conductive polymer has:a poly(ethylenedioxythiophene) backbone; a set of first functionalgroups pendant from the poly(ethylenedioxythiophene) backbone, whereinthe first functional groups comprise free succinimide groups, freemaleimide groups, or a combination thereof; and a set of secondfunctional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the second functional groups increase the solubilityof the polymer in water and act as electrical conductivity-enhancingdopants.

The methods functionalize a biological substrate having free aminegroups with a polymer that includes: a poly(ethylenedioxythiophene)backbone; a set of first functional groups pendant from thepoly(ethylenedioxythiophene) backbone, wherein the first functionalgroups comprise free succinimide groups, free maleimide groups, or acombination thereof; and a set of second functional groups pendant fromthe poly(ethylenedioxythiophene) backbone, wherein the second functionalgroups increase the solubility of the polymer in water. One embodimentof such a method includes the steps of: reacting the free succinimidegroups, the free maleimide groups, or both, of the polymer with the freeamine groups of the biological substrate, thereby covalently bonding thepolymer to the biological substrate.

One embodiment of a functionalized biological substrate includes: abiological substrate; a polymer having a poly(ethylenedioxythiophene)backbone; a set of first functional groups pendant from thepoly(ethylenedioxythiophene) backbone, wherein the first functionalgroups comprise free succinimide groups, free maleimide groups, or acombination thereof; and a set of second functional groups pendant fromthe poly(ethylenedioxythiophene) backbone, wherein the second functionalgroups increase the solubility of the polymer in water. The polymer iscovalently bonded to the biological substrate by covalent bonds formedby reactions between the succinimide groups of the polymer and aminegroups of the biological substrate.

One embodiment of a method of growing biological tissue includes thesteps of seeding a functionalized biological substrate and culturing theseeded functionalized biological substrate in a cell culture medium. Thefunctionalized biological substrate includes: a biological substrate; apolymer having a poly(ethylenedioxythiophene) backbone; a set of firstfunctional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the first functional groups comprise free succinimidegroups, free maleimide groups, or a combination thereof; and a set ofsecond functional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the second functional groups increase the solubilityof the polymer in water. The polymer is covalently bonded to thebiological substrate by covalent bonds formed by reactions between thesuccinimide groups of the polymer and amine groups of the biologicalsubstrate.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention will hereafter be describedwith reference to the accompanying drawings.

FIG. 1 shows a reaction scheme for the synthesis of a conductingconjugated polymer. The synthesis can be carried out without the use ofextremely oxidizing or acidic conditions, and, therefore, is suitablefor functionalizing allograft tissue. Palladium (Pd) catalysis can beused to make a co-polymer with more controlled stoichiometry, relativeto that provided by oxidative polymerizations. The conducting polymercan be oxidized after polymerization to dope it for enhanced electricalconductivity.

FIG. 2 illustrates covalent grafting of a conducting conjugated polymerto a collagen-containing substrate by simply soaking thecollagen-containing substrate in a PBS solution of the polymer. Graftingcan be carried out on both allograft tissue (including, but not limitedto, demineralized bone matrix (DBM) and acellular nerve grafts) andcollagen sponges, as demonstrated in the Example. R represents thependant sulfate group with organic linker shown in FIG. 1.

FIG. 3A and FIG. 3B show the results of functionalizing collagen spongeswith the CP of FIG. 2. FIG. 3A shows the viability of L929 fibroblastcells after 24 hours as a function of CP loading on the sponges. FIG. 3Bshows the loading of the L929 fibroblasts as a function of spongesoaking time. The loading began to level off after about 21 hours. Thefunctionalized sponges were then digested with collagenase, after whichthe CP remained water soluble.

FIGS. 4A and 4B show the live/dead staining results on rat BMSC cells onabsorbable collagen sponges (ACSs). The sponges had been soaked in asolution of the CP of FIG. 2 for one hour. FIG. 4A was taken after 24hours in culture. FIG. 4B was taken after seven days in culture. FIG. 4Cshows the sample fixed and stained with DRAQ5, demonstrating ideal MSCmorphology. FIG. 4D is an Alamar blue assay of the sample demonstratingcell viability for a CP-functionalized ACS similar to that provided byan unfunctionalized ACS, as a control.

FIGS. 5A and 5B show the up-regulation of some osteogenic-relevant geneson the CP-functionalized ACS (here the CP is referred to as PEDOT forsimplicity) in both osteogenic and standard media after one week (FIG.5A) and three weeks (FIG. 5B). In the graphs: ACS SM: Control instandard media; ACS/PEDOT SM: Functionalized material in standard media;ACS OM: Control in osteogenic media; and ACS/PEDOT OM: Functionalizedmaterial in osteogenic media.

DETAILED DESCRIPTION

Conjugated, electrically conducting polymers (CPs) with the ability tocovalently graft onto collagen are provided. Also provided are methodsof functionalizing biological tissues and other biological substrateswith the CPs, and methods of using the functionalized biologicalsubstrates as cell and tissue growth scaffolds that harness the passivetherapeutic benefits of CPs and use the enhanced conductivity providedby the scaffolds to stimulate cell growth and proliferation through thebulk of the biological substrate.

The described CPs are biocompatible water soluble, conjugated polymersthat allow for covalent attachment to a biological substrate viareactions with reactive functional groups on the substrate under mildconditions. For use in tissue growth and/or cell culture applications,the CPs are also cytocompatible. Use of a water-soluble conjugatedpolymer is particularly advantageous because it allows for renalclearance of the conducting element in vivo. As used herein, the termbiocompatible refers to a material that does not have a significantnegative impact on tissue growth and viability and/or a material that,if implanted in a living biological entity (e.g., a mammal, such as ahuman), does not cause an adverse reaction in that biological entity. Asused herein, the term cytocompatible refers to a material that isbiocompatible and, more specifically, that does not have an adverseeffect on the growth and viability of biological cells.

The CPs are characterized by: a poly(ethylenedioxythiophene) backbone; aset of first functional groups pendant from thepoly(ethylenedioxythiophene) backbone, wherein the first functionalgroups comprise chemical groups that react with a biological substrateto covalently graft the CPs to a the biological substrate; and a set ofsecond functional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the second functional groups increase the watersolubility of the CP. For the purposes of this disclosure a functionalgroup is considered to increase the water solubility of the CP if a CPhaving the pendant functional groups is more soluble in water than a CPthat lacks the pendant functional groups, but, otherwise, has the samestructure. In some embodiments, the first functional groups comprisefree succinimide groups, free maleamide groups, free thiol groups, or acombination of two or more thereof. In some embodiments, the secondfunctional groups comprise sulfate groups. Thepoly(ethylenedioxythiophene) backbone may consist of onlyethylenedioxythiophene units. As used herein, the term “free” indicatesthat the functional group is reactive and can form a covalent bond. Byway of illustration, one example of a CP comprises free succinimidegroups to impart the CP with covalent grafting ability and sulfategroups to impart water solubility to the CP.

The first and second functional groups can be attached to the polymerbackbone by organic linkers comprising carbon and hydrogen and,optionally, nitrogen, oxygen, and/or sulfur atoms. The organic linkersand pendant groups can be incorporated into a polymer using reactiveorganic linker molecules. The organic linker molecules include areactive group that reacts with the monomers that make up the CPbackbone or that reacts with the CP backbond itself and a secondreactive group that reacts with the first or second functional groups inorder to covalent attached those functional groups to the CP. The firstand second reactive groups may be separated by a linker chain. Thelinker chain may be a substituted or unsubstituted alkyl chain, asubstituted or unsubstituted alkenyl chain, or a substituted orunsubstituted alkynyl chain. By way of illustration, substituted orunsubstituted C₁-C₆ alkyl, alkenyl, or alkynyl chains may be used as thelinker chain. Substituted or unsubstituted heteroalkyl, heteroalkenyl,or heteroalkynyl chains can also be used as linker chains, whereheteroalkyl, heteroalkenyl, or heteroalkynyl chains refer to alkyl,alkenyl, and alkynyl chains (including C₁-C₆ alkyl, alkenyl, or alkynylchains), respectively, in which the carbon chain is interrupted by oneor more heteroatoms, such as nitrogen, oxygen, and/or sulfur atoms.Alkoxy chains are illustrative examples of heteroalkyl chains. In someexamples of the conductive polymers, the functional groups pendant fromthe electrically conductive polymers consist of only the first andsecond functional groups. This includes examples in which theside-chains extending from the polymer backbone consist of only thelinker chains and first and second functional groups listed herein.

One embodiment of a CP has the structure:

where A represents:

and B represents:

and n and m represent the number of repeat units for the respectiveunits in the polymer backbone. As illustrated by the CP above, thepolymers may be oxidized in order to dope them with charge-balancingcounterions, which enhance the electrical conductivity of the CP. In thedoped CPs the water solubility enhancing group has a negative change andacts as a counterion dopants that stabilize the positive charges on thepolymer backbone. By enhanced electrical conductivity, it is meant thatthe doped polymers are more electrically conductive than a polymer thatlacks the water solubility enhancing functional groups, but is otherwisethe same. Methods for making the CP shown above are described in theExample and illustrated in FIG. 1.

The synthesis of the CPs can be carried out using a Pd catalysis to makea co-polymer with more controlled stoichiometry, relative to thatprovided by oxidative polymerizations. This can be carried out withoutthe use of extremely oxidizing or acidic conditions, and, therefore, issuitable for functionalizing allograft tissue. The ratio of the monomersbearing the water solubility increasing groups (for example, sulfategroups) to the monomers bearing surface grafting groups (for example,succinimide groups) will vary depending on the desired level of watersolubility and the desired strength of surface bonding. Generally,however, the CPs will contain a greater number of the monomers bearingthe water solubility-increasing groups. By way of illustration, invarious embodiments of the CPs, the mole ratio of first functionalgroups to second functional groups is in the range from about 1:1 toabout 1:100. This includes embodiments in which the mole ratio of firstfunctional groups to second functional groups is in the range from about1:2 to 1:20 and further includes embodiments in which the mole ratio offirst functional groups to second functional groups is in the range fromabout 1:5 to 1:15.

The substrate grafting groups on the CPs can react with various reactivefunctionalities on a biological substrate to form covalent bonds to thesubstrate. Suitable reactive functionalities include, for example,nucleophilic groups, such as free amine groups and free hydroxy groups.By way of illustration, succinimide groups and maleimide groups canreact with free amine groups on a biological substrate having free aminegroups to form covalent bonds to the substrate. This can be accomplishedby infusing the biological substrate with the polymer in the presence ofphosphate buffer saline (PBS), as illustrated in the Example and shownin the upper panel of FIG. 2.

The biological substrates to which the CPs can be grafted are substratesthat are composed of biomolecules (e.g., biological tissues) or thatincorporate biomolecules into their structures. For example, thebiomolecules may be proteins, such as collagen, which has free aminesand is a primary component of extracellular matrix (ECM). This issignificant because decellularized ECM (dECM) harvested from humans andother animals preserves the macro- and micro-structure of the tissuesfrom which it is harvested and, thus, has found a wide range of bothclinical and pre-clinical applications.

The biohybrid composites that result from grafting the CPs onto abiological substrate have augmented electroactivity, which facilitatesin vitro cell proliferation and enables bulk electrical stimulation toenhance tissue regeneration. The tissue growth or regeneration may bepassive—that is, without the application of an external electrical bias,or active—that is, carried out under the influence of an appliedelectrical bias. Electrical recording devices and/or electricalstimulation devices can be integrated directly into the biologicalsample to monitor and/or promote tissue growth.

Moreover, when the CP material binds to native tissues, such ascollagen-containing human allograft, the native tissue architecture andpresence of native bioactive molecules, such as growth factors, remainundisturbed, which renders the functionalized tissue well-suited for useas allograft implants.

The CP-grafted biological substrates can be used as biological cell andtissue growth scaffolds by seeding the functionalized biologicalsubstrates with biological cells, such as human mesenchymal cells(hMSCs), and culturing the seeded biological substrates in a cellculture medium, including a differentiation medium or a proliferationmedium. Alternatively, the CP-grafted substrates can be implanted intoan animal (e.g., human) to enable in vivo cell growth, differentiation,and proliferation. Tissues that can be grown using the tissue growthscaffolds include dermal, neural, osteo, chondral, cardiac, andosteochondral tissue.

Example

A conducting conjugated polymer was synthesized according the reactionscheme shown in FIG. 1. A Pd catalysis was used to make a co-polymerwith controlled stoichiometry and the conducting polymer was oxidizedafter polymerization to dope it for enhanced conductivity.

The conducting conjugated polymer was grafted to a collagen-containingsubstrate by simply soaking the collagen-containing substrate in a PBSsolution of the polymer, as shown in FIG. 2. Grafting was carried out onDBM and acellular collagen sponges.

L929 fibroblast cells were cultured on the acellular collagen spongesgrafted with the CP of FIG. 2. FIG. 3A shows the viability of L929fibroblast cells after 24 hours as a function of CP loading on thesponges. FIG. 3B shows the loading of the L929 fibroblasts as a functionof sponge soaking time. The loading began to level off after about 21hours. The functionalized sponges were then digested with collagenase,after which the CP remained water soluble.

Rat BMSC cells were also cultures on the acellular collagen spongesgrafted with the CP of FIG. 2. FIGS. 4A and 4B show the live/deadstaining results for the rat BMSC cells on the absorbable collagensponges. The sponges had been soaked in a solution of the CP of FIG. 2for one hour. FIG. 4A was taken after 24 hours in culture. FIG. 4B wastaken after seven days in culture. FIG. 4C shows the sample fixed andstained with DRAQ5, demonstrating ideal MSC morphology. FIG. 4D is anAlamar blue assay of the sample demonstrating cell viability for aCP-functionalized ACS similar to that provided by an unfunctionalizedACS, as a control.

The up-regulation of some osteogenic-relevant genes on theCP-functionalized ACS (here the CP is referred to as PEDOT forsimplicity) was measured in both osteogenic and standard media after oneweek (FIG. 5A) and three weeks (FIG. 5B). In the graphs: ACS SM: Controlin standard media; ACS/PEDOT SM: Functionalized material in standardmedia; ACS OM: Control in osteogenic media; and ACS/PEDOT OM:Functionalized material in osteogenic media.

The word “illustrative” is used herein to mean serving as an example,instance, or illustration. Any aspect or design described herein as“illustrative” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Further, for the purposes ofthis disclosure and unless otherwise specified, “a” or “an” can mean“only one” or can mean “one or more.”

The foregoing description of illustrative embodiments of the inventionhas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of theinvention. The embodiments were chosen and described in order to explainthe principles of the invention and as practical applications of theinvention to enable one skilled in the art to utilize the invention invarious embodiments and with various modifications as suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and theirequivalents.

What is claimed is:
 1. A biocompatible electronically conductive polymercomprising: a poly(ethylenedioxythiophene) backbone; a set of firstfunctional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the first functional groups comprise free succinimidegroups, free maleimide groups, or a combination thereof; and a set ofsecond functional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the second functional groups increase the solubilityof the polymer in water and act as electrical conductivity-enhancingdopants.
 2. The biocompatible polymer of claim 1, wherein the secondfunctional groups comprise sulfate groups.
 3. The biocompatible polymerof claim 2, wherein the first functional groups comprise the freesuccinimide groups.
 4. The biocompatible polymer of claim 3, wherein thesuccinimide groups and the sulfate groups are covalently bonded toethylenedioxythiophene groups in the backbone via organic linkers. 5.The biocompatible polymer of claim 4, wherein the organic linkerscomprise a substituted or unsubstituted alkyl chain, a substituted orunsubstituted heteroalkyl chain, or a combination thereof.
 6. Thebiocompatible polymer of claim 5, having the structure:

where A represents:

and B represents:

and n and m represent the number of repeat units in the polymerbackbone.
 7. The biocompatible polymer of claim 3, wherein the moleratio of free succinimide groups to sulfate groups is in the range from1:1 to 1:100.
 8. The biocompatible polymer of claim 3, wherein the moleratio of free succinimide groups to sulfate groups is in the range from1:5 to 1:15.
 9. The biocompatible polymer of claim 1, wherein the moleratio of first functional groups to second functional groups is in therange from 1:1 to 1:100.
 10. The biocompatible polymer of claim 1,wherein the polymer is cytocompatible.
 11. A method of functionalizing abiological substrate comprising free amine groups with a polymercomprising: a poly(ethylenedioxythiophene) backbone; a set of firstfunctional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the first functional groups comprise free succinimidegroups, free maleimide groups, or a combination thereof; and a set ofsecond functional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the second functional groups increase the solubilityof the polymer in water, the method comprising reacting the freesuccinimide groups, the free maleimide groups, or both, of the polymerwith the free amine groups of the biological substrate, therebycovalently bonding the polymer to the biological substrate.
 12. Themethod of claim 11, wherein the second functional groups comprisesulfate groups.
 13. The method of claim 12, wherein the first functionalgroups comprise the free succinimide groups.
 14. The method of claim 13,wherein the succinimide groups and the sulfate groups are covalentlybonded to ethylenedioxythiophene groups in the backbone via organiclinkers.
 15. The method of claim 14, wherein the organic linkerscomprise a substituted or unsubstituted alkyl chain, a substituted orunsubstituted heteroalkyl chain, or a combination thereof.
 16. Themethod of claim 13, wherein the mole ratio of free succinimide groups tosulfate groups is in the range from 1:1 to 1:100.
 17. The method ofclaim 13, wherein the mole ratio of free succinimide groups to sulfategroups is in the range from 1:5 to 1:15.
 18. A functionalized biologicalsubstrate comprising: a biological substrate; and a polymer comprising:a poly(ethylenedioxythiophene) backbone; a set of first functionalgroups pendant from the poly(ethylenedioxythiophene) backbone, whereinthe first functional groups comprise free succinimide groups, freemaleimide groups, or a combination thereof; and a set of secondfunctional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the second functional groups increase the solubilityof the polymer in water, wherein the polymer is covalently bonded to thebiological substrate by covalent bonds formed by reactions between thesuccinimide groups of the polymer and amine groups of the biologicalsubstrate.
 19. The functionalized biological substrate of claim 18,wherein the biological substrate comprises collagen.
 20. A method ofgrowing biological tissue, the method comprising: seeding afunctionalized biological substrate comprising: a biological substrate;and a polymer comprising: a poly(ethylenedioxythiophene) backbone; a setof first functional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the first functional groups comprise free succinimidegroups, free maleimide groups, or a combination thereof; and a set ofsecond functional groups pendant from the poly(ethylenedioxythiophene)backbone, wherein the second functional groups increase the solubilityof the polymer in water, wherein the polymer is covalently bonded to thebiological substrate by covalent bonds formed by reactions between thesuccinimide groups of the polymer and amine groups of the biologicalsubstrate; and culturing the seeded functionalized biological substratein a cell culture medium.